In vitro proliferation and differentiation of erythroid progenitors from patients with myelodysplastic syndromes: evidence for Fas-dependent apoptosis

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1594-1601 ◽  
Author(s):  
Yann-Erick Claessens ◽  
Didier Bouscary ◽  
Jean-Michel Dupont ◽  
Françoise Picard ◽  
Josiane Melle ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Liquid Culture ◽  
Fas Ligand ◽  
Chimeric Protein ◽  
Refractory Anemia ◽  
Erythroid Progenitors ◽  
In Vitro Proliferation ◽  
Cd34 Cells ◽  
Proliferation And Differentiation

Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34+ cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulinlike growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 103fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34+ cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34+-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.

GATA-1 Overexpression Does Not Restore Deficient Erythropoiesis in Myelodysplastic Syndromes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3430-3430
Author(s):  
Alexandra Rideau ◽  
Stephane Durual ◽  
Maciej Wiznerowics ◽  
Sylvie Ruault ◽  
Vincent Piguet ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Culture System ◽  
Cell Cycle Control ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Differentiation Markers ◽  
Cd34 Cells ◽  
Erythroleukemic Cell

Abstract Introduction: Transcription factor GATA-1 is essential for erythroid and megakaryocytic maturation. A role of GATA-1 in cell cycle control is suggested by the fact that GATA-1 mutations are associated with hematopoietic precursor proliferation and leukemogenesis and that defective GATA-1 expression is observed in in vitro cultures of erythroid myelodysplastic precursors. In order to study more in detail a potential role of GATA-1 dysregulation in myelodysplastic syndromes (MDS), we constructed lentiviral vectors with the aim to overexpress GATA-1 protein or to inhibit its production in erythroid progenitors. Methods and Results: Using RNA interference technology we tested how GATA-1 inhibition interfered with erythroid differentiation. We selected one GATA-1 specific siRNA, which abolished expression of GATA-1 protein in K562 and HEL erythroleukemic cell lines, as verified by Western blot. Interestingly, we observed in parallel to the disappearance of GATA-1 protein, decreased proliferation rates (170x for K562 and 30x for HEL after 17 days of culture) and increased apoptosis. Normal CD34+ cells cultured in our culture system and transduced with the siRNA vector were practically blocked in their erythroid differentiation: 14 % glyco+/CD36- mature erythroid cells versus 81 % in untransduced and 80 % in cultures transduced with control lentivector (obtained after 14 days of culture). Differentiation into myeloid cells was not affected. To overexpress GATA-1 we cloned the wild-type as well as a mutated, caspase-resistant, form of GATA-1 in a pWPIR-ires-GFP bicistronic lentivector. Functionality of both lentivectors was validated in HeLa cells. For the study of GATA-1 in primary human hematopoietic cells we used an in vitro culture system in which CD34+ progenitors differentiate into mature red blood cells in the presence of erythropoietin, IL-3, and SCF. Transduction of CD34+ cells with lentivectors led to increase of GATA-1 mRNA (400-fold) measured by Realtime RT-PCR and to detection of protein. No difference was observed in cell numbers, expression of erythroid differentiation markers and survival between cells transduced with control vector and with pWPIR-GATA-1-ires-GFP. CD34+ cells from 3 patients with low-risk MDS in this culture system proliferated less (15x ± 13 amplification after 14 days of culture versus 72x ± 35 for normal precursors) differentiated less, and became apoptotic earlier than normal cells. However, overexpression of GATA-1 did not restore proliferation rate, nor did it lead to increased erythroid differentiation, or increase in survival. Conclusion: GATA-1 overexpression was not able to overcome defective erythroid differentiation of myelodysplastic progenitors, nor did it increase differentiation of normal erythroid progenitors. On the other hand, GATA-1 inhibition in normal erythroid precursors led to blockage of erythroid differentiation. We therefore assume that either factors upstream of GATA-1 or additional, GATA-1 independent factors, are responsible for the myelodysplastic phenotype.

IL-8 and its CXCR1 and CXCR2 receptors participate in the control of megakaryocytic proliferation, differentiation, and ploidy in myeloid metaplasia with myelofibrosis

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 464-473 ◽  
Author(s):  
Sharareh Emadi ◽  
Denis Clay ◽  
Christophe Desterke ◽  
Bernadette Guerton ◽  
Eliane Maquarre ◽  
...  
Keyword(s):  
Liquid Culture ◽  
Potential Role ◽  
Culture Conditions ◽  
Hematopoietic Progenitor ◽  
Myeloid Metaplasia ◽  
Incurable Disease ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
In Vitro Inhibition

AbstractMyeloproliferation, myelofibrosis, and neoangiogenesis are the 3 major intrinsic pathophysiologic features of myeloid metaplasia with myelofibrosis (MMM). The myeloproliferation is characterized by an increased number of circulating CD34+ progenitors with the prominent amplification of dystrophic megakaryocytic (MK) cells and myeloid metaplasia in the spleen and liver. The various biologic activities of interleukin 8 (IL-8) in hematopoietic progenitor proliferation and mobilization as well as in neoangiogenesis prompted us to analyze its potential role in MMM. We showed that the level of IL-8 chemokine is significantly increased in the serum of patients and that various hematopoietic cells, including platelets, participate in its production. In vitro inhibition of autocrine IL-8 expressed by CD34+ cells with either a neutralizing or an antisense anti–IL-8 treatment increases the proliferation of MMM CD34+-derived cells and stimulates their MK differentiation. Moreover, addition of neutralizing anti–IL-8 receptor (CXC chemokine receptor 1 [CXCR1] or 2 [CXCR2]) antibodies to MMM CD34+ cells cultured under MK liquid culture conditions increases the proliferation and differentiation of MMM CD41+ MK cells and restores their polyploidization. Our results suggest that IL-8 and its receptors participate in the altered MK growth that features MMM and open new therapeutic prospects for this still incurable disease.

In Vitro Proliferation and Differentiation of Human Hair Follicle Stem Cells on Chitosan-Skin Regenerating Template

2015 ◽  
Vol 05 (999) ◽  
pp. 1-1
Author(s):  
Abu Bakar Mohd Hilmi ◽  
Mohd Noor Norhayati ◽  
Ahmad Sukari Halim ◽  
Chin Keong Lim ◽  
Zulkifli Mustafa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hair Follicle ◽  
Human Hair ◽  
Human Hair Follicle ◽  
In Vitro Proliferation ◽  
Proliferation And Differentiation ◽  
Hair Follicle Stem Cells ◽  
Follicle Stem Cells

scholarly journals Myeloid differentiation of purified CD34+ cells after stimulation with recombinant human granulocyte-monocyte colony-stimulating factor (CSF), granulocyte-CSF, monocyte-CSF, and interleukin-3

Blood ◽  
1991 ◽  
Vol 78 (12) ◽  
pp. 3192-3199 ◽  
Author(s):  
T Egeland ◽  
R Steen ◽  
H Quarsten ◽  
G Gaudernack ◽  
YC Yang ◽  
...  
Keyword(s):  
Myeloid Differentiation ◽  
Cell Multiplication ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Stimulating Factor

Abstract CD34+ cells isolated from bone marrow or umbilical cord blood from healthy donors were studied for proliferation and differentiation in liquid cultures in the presence of recombinant human granulocyte- monocyte colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), monocyte CSF (M-CSF), and interleukin-3 (IL-3), followed by immunophenotyping for myeloid and myeloid-associated cell surface markers. IL-3, either alone or together with GM-CSF, G-CSF, or M-CSF, induced, on average, 50-fold cell multiplication, GM-CSF five fold to 10-fold, and G-CSF and M-CSF less than fivefold. Cells from cultures stimulated with GM-CSF, G-CSF, or M-CSF alone contained cells with a “broad” myeloid profile, “broader” than observed in cultures with IL-3. However, since IL-3 induced rapid cell multiplication, high numbers of cells expressing early (CD13, CD33) and late myeloid markers (CD14, CD15) were recovered. The presence of other CSFs together with IL-3 did not alter the IL-3-induced effect on the cells. When 5,000 CD34+ cells were cultured with IL-3 alone, the cultures still contained 2,000 to 5,000 CD34+ cells after 14 days of culture, while cells cultured with GM-CSF, G-CSF, or M-CSF contained less than 1,000 CD34+ cells. Furthermore, 1,000 to 3,000 cells were positive for the megakaryocytic lineage marker CD41b after cultures with GM-CSF or IL-3, while cultures with G-CSF or M-CSF did not contain detectable numbers of CD41b+ cells. Finally, erythroid cells could also be generated from purified CD34+ cells. The results show that IL-3 and GM-CSF can induce rapid proliferation of purified CD34+ cells in vitro with differentiation to multiple myeloid lineages, while certain subsets maintain expression of CD34.

scholarly journals Effects of recombinant human interleukin-3 on CD34-enriched normal hematopoietic progenitors and on myeloblastic leukemia cells

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1580-1588 ◽  
Author(s):  
S Saeland ◽  
C Caux ◽  
C Favre ◽  
JP Aubry ◽  
P Mannoni ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Erythroid Progenitors ◽  
Phenotypic Analysis ◽  
Proliferating Cells ◽  
Interleukin 3 ◽  
Cd34 Antigen ◽  
Cd34 Cells ◽  
Proliferation And Differentiation

Abstract Induction of proliferation and differentiation in response to recombinant human interleukin-3 (hIL-3) was studied in liquid and semisolid cultures of umbilical cord blood and bone marrow cells that were fractionated by “panning” with anti-My10 antibody according to expression of CD34 antigen. Cells from enriched fractions (70% to 90% CD34+) were found to proliferate strongly in response to hIL-3. Phenotypic analysis and morphologic characterization of the proliferating cells demonstrated a rapid decrease in CD34+ cells and an exponential increase in the number of cells belonging to the neutrophilic, eosinophilic, monocyte/macrophage, and thrombocytic lineages. When combined with recombinant human erythropoietin, burst colonies and cells expressing glycophorin-A were detected, thereby demonstrating the effects of hIL-3 on erythroid progenitors. Further, the development of mixed-erythroid colonies indicated that multipotential cells within CD34-enriched fractions responded to hIL-3. In addition, we examined the effect of hIL-3 on the proliferation of primary acute myeloblastic leukemia cells in liquid culture. We found that hIL-3 was able to induce cell proliferation in a proportion of the cases tested. Heterogeneity of the responses to hIL-3 was in part related to French-American-British classification but could not be correlated with CD34 antigen expression by the leukemic cells. These results indicate that, although the effects of hIL-3 on proliferation and differentiation of cells obtained from normal hematopoietic specimens were primarily borne by CD34+ cells, expression of the CD34 molecule per se is an insufficient condition to determine a growth response to this lymphokine.

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